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EP2643484A2 - Procédés d'identification d'un transcrit cellulaire naissant d'arn - Google Patents

Procédés d'identification d'un transcrit cellulaire naissant d'arn

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Publication number
EP2643484A2
EP2643484A2 EP11844470.2A EP11844470A EP2643484A2 EP 2643484 A2 EP2643484 A2 EP 2643484A2 EP 11844470 A EP11844470 A EP 11844470A EP 2643484 A2 EP2643484 A2 EP 2643484A2
Authority
EP
European Patent Office
Prior art keywords
cell
transcription
transcript
sequencing
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11844470.2A
Other languages
German (de)
English (en)
Other versions
EP2643484A4 (fr
Inventor
Jonathan Weissman
Lee Stirling Churchman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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Publication date
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP2643484A2 publication Critical patent/EP2643484A2/fr
Publication of EP2643484A4 publication Critical patent/EP2643484A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • RNA polymerase inhibitors include, e.g., small molecules that inhibit RNA polymerase or interact with DNA to block transcription.
  • RNA polymerase inhibitors include actinomycin D, which intercalates into double stranded DNA and blocks the movement of RNA polymerase and rifampicin, an antibiotic which binds the ⁇ subunit of RNA polymerase and blocks initiation of transcription.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the RNA polymerase complex.
  • the preferred salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins.
  • His-tag e.g., 6x histidine
  • MBP maltose binding protein
  • GST glutthione-S-transferase
  • the cellular nascent RNA transcript can be sequenced using tools and method well- known to those of skill in the art. For example, a direct sequencing technique may be employed (e.g., Sanger sequencing). In some embodiments, a deep sequencing technique is used. Certain embodiments employ sequencing platforms of Illumina, Inc. (San Diego, Calif.) and/or 454 Corporation (Roche Diagnostics, Basel, Switzerland), e.g., the Genome Sequencer FLX System, which employs pyrosequencing to provide long read lengths and very high single-read accuracy. In some embodiments, other sequencing platforms are utilized, including, but not limited to OmniMoRA (Reveo, Inc. (Elmsford, N.Y.)), VisiGen® (VisiGen Biotechnologies, Inc.
  • OmniMoRA Reveo, Inc. (Elmsford, N.Y.)
  • VisiGen® VisiGen Biotechnologies, Inc.
  • the invention is not limited to any particular methodology or product for analyzing the cellular nascent RNA transcript.
  • “pyrosequencing,” as known in the art, refers to a method of sequencing by synthesis which relies on detection of pyrophosphate release on nucleotide incorporation. See e.g., Ronaghi et al, Science 1998, 281:363; Ronaghi et al, Anal. Biochem. 242:84; Nyren et al, Methods Mol. Biology., 2007, 373: 1-14.
  • the term “sequencing by ligation” refers to a DNA sequencing method that uses DNA ligase, as known in the art, to identify the nucleotide present at a given position in a DNA sequence.
  • the term “sequencing by extension” refers to a DNA sequencing method wherein a primer is extended with a known or detectable nucleotide, as known in the art.
  • the present methods can be used in conjunction with a variety of sequencing techniques.
  • the process to determine the nucleotide sequence can be an automated process.
  • the illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M.J. et al.
  • SMRT real-time DNA sequencing technology
  • Pacific Biosciences Inc. can be utilized with the methods described herein.
  • a SMRT chip or the like may be utilized (U.S. Patent Nos. 7,181,122, 7,302,146, 7,313,308, incorporated by reference in their entireties and for all purposes).
  • a SMRT chip comprises a plurality of zero-mode waveguides (ZMW). Each ZMW comprises a cylindrical hole tens of nanometers in diameter perforating a thin metal film supported by a transparent substrate.
  • Attenuated light may penetrate the lower 20-30 nm of each ZMW creating a detection volume of about 1 x 10-21 L. Smaller detection volumes increase the sensitivity of detecting fluorescent signals by reducing the amount of background that can be observed.
  • the poly(T) oligonucleotide can be used as a primer for the extension of a polynucleotide complementary to the target nucleic acid.
  • fluorescently-labeled nucleotide monomer namely, A, C, G, or T
  • Incorporation of a labeled nucleotide into the polynucleotide complementary to the target nucleic acid is detected, and the position of the fluorescent signal on the glass cover slip indicates the molecule that has been extended.
  • the fluorescent label is removed before the next nucleotide is added to continue the sequencing cycle. Tracking nucleotide incorporation in each polynucleotide strand can provide sequence information for each individual target nucleic acid.
  • Target nucleic acids e.g., the cellular nascent RNA transcripts
  • target nucleic acid sequences are interspersed approximately every 20 bp with adaptor sequences.
  • the target nucleic acids can be amplified using rolling circle replication, and the amplified target nucleic acids can be used to prepare an array of target nucleic acids.
  • Methods of sequencing such arrays include sequencing by ligation, in particular, sequencing by combinatorial probe-anchor ligation (cPAL).
  • a pool of probes that includes four distinct labels for each base is used to read the positions adjacent to each adaptor.
  • a separate pool is used to read each position.
  • a pool of probes and an anchor specific to a particular adaptor is delivered to the target nucleic acid in the presence of ligase.
  • the anchor hybridizes to the adaptor, and a probe hybridizes to the target nucleic acid adjacent to the adaptor.
  • the anchor and probe are ligated to one another. The hybridization is detected and the anchor-probe complex is removed.
  • a different anchor and pool of probes is delivered to the target nucleic acid in the presence of ligase.
  • the sequencing methods described herein can be advantageously carried out in multiplex formats such that multiple different target nucleic acids (e.g., the cellular nascent RNA transcripts) are manipulated simultaneously.
  • different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner.
  • the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner.
  • the target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface.
  • the array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail herein.
  • bridge in this context refers to the fact that during the annealing step, the extension product from one bound primer forms a bridge to the other bound primer. All amplified products are covalently bound to the surface, and can be detected and quantified without electrophoresis. Sequencing by synthesis methods may be employed with any appropriate amplification method, including for example PCR. In some embodiments, the sequencing is accomplished using deep sequencing,
  • nucleic acid molecules e.g., the cellular nascent RNA transcript
  • nucleic acid molecules can be amplified on beads, for example using emulsion PCR methods.
  • Exemplary emulsion- based amplification techniques that can be used in a method disclosed herein are described in US 2005/0042648; US 2005/0079510; US 2005/0130173 and WO 05/010145, each of which is incorporated herein by reference in its entirety and for all purposes.
  • nucleic acid molecules can be amplified on a surface using bridge amplification to form nucleic acid clusters. Exemplary methods of generating nucleic acid clusters for use in high-throughput nucleic acid technologies have been described.
  • the stimulus is a small molecule (e.g., a small molecule drug).
  • Transcriptional activity may be determined in the presence or absence of introduced small molecules.
  • suitable small molecules suitable for the methods of the invention include chemicals from a chemical library or molecules identified from natural product screening.
  • the stimulus is an inhibitory nucleic acid.
  • Transcriptional activity may be determined in the presence or absence of introduced inhibitory nucleic acids.
  • Inhibitory nucleic acids suitable for the methods of the invention include genetic molecules such as co-suppression or antisense molecules or RNAi-inducing molecules (e.g., siR A, shRNA, ribozymes).
  • antisense molecules or RNAi-inducing molecules are from about 10 base pairs long to about 2000 base pairs long, or from about 12 to about 30 base pairs long such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, base pairs in length. In some embodiments, the antisense molecule or RNAi-inducing molecule is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 base pairs in length.
  • the antisense molecule or RNAi-inducing molecule is about 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 base pairs in length. In some embodiments, the antisense molecule or RNAi-inducing molecule (e.g.
  • co- suppression molecules include double-stranded RNA molecules forming a hairpin with or without single-stranded portions in the form of a "bulge" or "bubble".
  • Inhibitory nucleic acids can be delivered to a cell by direct transfection (e.g., using liposomal transfection reagents) or transfection and expression via an expression vector (e.g., a mammalian expression vector or a viral expression vector).
  • the molecules suitable for the methods of the invention include molecules from a "combinatorial chemical library.”
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries are well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354:84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec.
  • another aspect of the present invention provides a method for determinin changes in transcriptional activity in a cell or cell linage.
  • the method involves identifying cellular nascent RNA transcripts in cells in the presence or absence of a stimulus, or cells at different developmental stages, or diseased cells and normal cells.
  • the method further involves arresting transcription in the cells described above, and purifying the native cellular nascent RNA transcripts from these cells (e.g., as part of an RNA polymerase complex).
  • these native cellular nascent RNA transcripts are then sequenced. Based on the sequences of the native cellular nascent RNA transcripts, the transcriptional activity in these cells are assessed and compared. For example, the changes in transcriptional activity in cells in the presence or absence of a stimulus are measured and compared. The differences in transcriptional activity in cells at different developmental stages are detected and compared. Further the transcriptional activity in normal cells and diseased cells can also be compared.
  • the methods of the invention disclosed herein can be used for various assays.
  • the methods described herein can be used to detect relative or absolute gene expression levels as indicated by cellular nascent RNA transcripts, or detect the relative or absolute amount of noncoding RNAs or unstable RNAs.
  • the methods described herein can be used to detect allelic expressions.
  • the methods described herein can be used for haplotype assays and phasing of multiple SNPs within chromosomes.
  • the methods described herein can be used to detect DNA methylation state, mRNA alternate splicing and level of splice variants. Further, the methods can be used to identify or detect the presence of microbe or viral content in food and
  • nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.
  • RNAPII protein-coding genes
  • a functional variant of RNAPII with a 3x-FLAG epitope attached to its third subunit (Rpb3).
  • Log- phase cultures were collected by filtration and flash frozen in liquid nitrogen (Fig. la). After cryogenic lysis, RNAPII was efficiently immunoprecipitated (Fig. 7a).
  • Fig. 7a We prepared the co- purified RNA for deep sequencing using a protocol that allows efficient RNA capture while minimizing bias (Ingolia, N. T.
  • Rpd3S This raises the question of how Rpd3S is recruited to positions designated for suppression of antisense transcription.
  • the Rcol and Eaf3 components of the Rpd3S complex bind H3 lysine 36 methylation marks made by Set2 and that binding activates Rpd3S's deacetylase activity (Carrozza, M. J. et al, Cell 123:581-592 (2005); Keogh, M. C. et al, Cell 123:593-605 (2005); Li, B. et al, J. Biol. Chem. 284:7970-7976 (2009)).
  • RNAPII RNAPII-associated methyltransferase
  • RNAP pausing has been shown to involve backtracking: after encountering a blockage, RNAP reverses direction and moves upstream (Nudler, E. et al, Cell 89:33-41 (1997)).
  • TFIIS elongation factor
  • Fig. 5a Izban, M. G. & Luse, D. S., J. Biol.
  • nucleosomes induce RNAPII backtracking and TFIIS aids the progression of RNAPII through them (Hodges, C. et al, Science 325:626-628 (2009); Kireeva, M. L. et al, Cell 18:97-108 (2005)).
  • chromatin remodelling factors could greatly diminish the nucleosome barrier or remove nucleosomes prior to RNAPII arrival (Li, B. et al, Cell 128:707-719 (2007); Petesch, S. J. & Lis, J. T., Cell 134:74- 84 (2008)).
  • sense transcription may also use this termination mechanism as our data showed an enrichment for transcripts at the 5 ' end of genes that mirrors what we observed for antisense transcripts and complements observations that Nrdl localizes to the 5' end of genes (Vasiljeva, L. et al, Nat. Struct. Mol. Biol. 15:795-804 (2008)).
  • our observations suggest an independence between the initiation of the sense and antisense transcripts. Specifically, we found only modest correlation between sense and antisense transcription levels. Moreover, even among the set of antisense transcripts that increased when RCOI is deleted, no increase in sense transcription levels was seen.
  • NET-seq provides the first in-depth view of pausing in a eukaryotic cell revealing that transcription is punctuated by pauses throughout the body of all messages. Taking into account both the abundance and magnitude of the pauses, we conclude that R APII spends comparable time in a paused state and moving forward (Fig. 16).
  • nucleosomes induce pausing in vivo, and may be the major source of pausing considering that the increase in pause density at nucleosomes is comparable to the increase in nucleosome occupancy (Weiner, A. et al, Genome Res 20:90-100 (2010)).
  • cDNA synthesis and sequencing was performed as described with a few modifications (Ingolia, N. T. et al, Science 324:218-223 (2009)).
  • the sequencing primer binding site was positioned so that sequencing would start at the 3 ' end.
  • nucleosome positions (Weiner, A. et al, Genome Res 20:90-100 (2010)) were assigned as +l,+2,+3, etc., according to their position relative to transcription start sites. The pause density relative to a particular nucleosome was determined by the number of pauses observed at that position divided by the total number of opportunities it could be observed there.
  • k is the nucleosome number
  • g(y) is the binary function indicating whether a pause occurs at y
  • « z k- are the centre nucleosome positions.
  • the error of the pause density was calculated via the standard deviation of the binomial distribution
  • the culture was scrapped off the filter with a spatula pre-chilled by -EN2 and flash frozen by plunging into -EN2. Frozen cells were pulverized for six cycles, each of 3 min. at 15 Hz, on a Retsch MM301 mixer mill. Sample chambers were pre-chilled in -EN2 and re-chilled between each pulverization cycle.
  • DNAse I Promega, RQl RNase-Free DNase
  • Bound proteins were eluted twice with 150 ⁇ elution buffer (20 mM Hepes, pH 7.4, 110 mM KOAc, 0.5% Triton, 0.1% Tween) with 2 mg ml "1 3 x Flag peptide (Sigma Aldrich).
  • RNA from the combined eluates was purified using the miRNeasy kit (Qiagen, 217004). A typical yield from approximately one liter of log- phase yeast culture was 3 ⁇ g.
  • RNA was purified by a standard isopropanol precipitation as follows. After adding 650 ⁇ , of isopropanol, samples were placed at -30°C for at least 30 minutes. Precipitated RNA was pelleted by centrifugation at 4°C at 20000xg for 30 minutes. The pellet was air dried after a quick wash with 80% ethanol and then resuspended in 10 mM Tris pH 7.0.
  • a DNA linker (SEQ ID NO: l 1) was ligated onto the 3 ' end of the immunoprecipitated RNA, the fragmented mRNA and a synthetic 28 base RNA oligonucleotide (oNTl 199, 5 '-AUGUACACGGAGUCGACCCGCA ACGCGA (SEQ ID NO:2)) similarly to what has been described (Unrau, P. J. & Bartel, D. P., Nature 395 :260-263 (1998)). Specifically, 3 ⁇ g of each RNA sample was broken into 3 reactions and diluted to 10 ⁇ ⁇ with 10 mM Tris, pH 7.0.
  • the ligated and fragmented samples were size-selected by gel electrophoresis.
  • the purified reactions along with the oNTI199 R A oligo was mixed with 2x No vex TBE-Urea sample prep buffer (Invitrogen) and briefly denatured, then loaded on a Novex denaturing 15% polyacrylamide TBE-urea gel (Invitrogen) and run according to the manufacturer's instructions.
  • the gel was stained with SYBR Gold (Invitrogen) and the 35-85 nt region was excised.
  • the gel was physically disrupted and either allowed to soak overnight in gel elution buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.1 U ⁇ 1 SUPERase'In) or incubated in 200 ⁇ of DEPC water for 10 min at 70°C.
  • gel elution buffer 300 mM NaOAc pH 5.5, 1 mM EDTA, 0.1 U ⁇ 1 SUPERase'In
  • the gel debris was removed from the water or buffer using a Spin-X column (Corning) and RNA was precipitated with GlycoBlue as a coprecipitant using standard methods.
  • the primer used for reverse transcription was oLSC003 (5'pTCGTATGCCGTCTTCTGCTTG (SEQ ID NO:3) ⁇
  • AATGATACGGCGACCACCGATCCGACGATCATTGATGGTGCCTACAG (SEQ ID NO:4)) where the initial p indicates 5' phosphorylation and ⁇ indicates a spacer, 18 carbon spacer-C ACTCA- 18 carbon spacer.
  • Efficient circularization of the RT product was performed as described (Ingolia, N. T. et al, Science 324:218-223 (2009)) with CircLigase (Epicentre) according to the manufacturer's directions. Any ligation bias at this step is averaged out as the random fragmentation leaves a range of 5 ' ends for each 3 ' end.
  • the PCR was performed directly on the circularized product as described (Ingolia, N. T.
  • DNA was purified from a PCR reaction that had not reached saturation and was quantified using the Agilent BioAnalyzer High Sensitivity DNA assay. DNA was then sequenced on the Illumina Genome Analyzer 2 according to the manufacturer's instructions, using 4 to 6 pM template for cluster generation and sequencing primer 0LSCOO6
  • 18 bp sequences would occur by chance every 6.9 e+ 10 bp which is sufficiently rare for 18 bp sequences to be generally uniquely aligned to the the 1.2 e+7 bp yeast genome. Alignments were first performed against tRNA and rRNA sequences to remove them. The remaining sequences were aligned against a recent version of the yeast genome downloaded from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/) on October 11 , 2009. Statistics on sequence alignments are reported in Table 1.
  • Metagene analysis Each gene included in the analysis is normalized by the mean number of reads in a 400 bp window beginning 100 bases downstream from the transcription start site. A mean read density (MRD) is then calculated for each position over all genes as described below.
  • MRD mean read density
  • i y n + x
  • k is the nucleosome number
  • g(y) is the binary function indicating whether a pause occurs
  • the densities were then binned by averaging across windows ten nucleotides wide.
  • the error for each bin was calculated by computing the sum of the variances of the binned measurements and calculating the square root.
  • nascent RNA was 0.34% of the total RNA. Alignments to genomic regions represented 27%>-42%> of our total reads, thus the IP provided an approximately 100-fold enrichment for nascent RNA consistent with the direct measurement of enrichment made in our mixed lysate experiment (Table 2).
  • TDH3:GFP ratio which is summarized in the table. As the first IP had half the amount of labeled Rpb3 than the second IP, these results show that messages expressed in the same cells as a labeled Rpb3 are purified at least 100-fold more than messages from other cells.

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Abstract

L'invention concerne des procédés et des compositions pour l'identification d'un transcrit cellulaire naissant d'ARN.
EP11844470.2A 2010-11-22 2011-11-22 Procédés d'identification d'un transcrit cellulaire naissant d'arn Withdrawn EP2643484A4 (fr)

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EP2643484A4 (fr) 2010-11-22 2014-04-16 Univ California Procédés d'identification d'un transcrit cellulaire naissant d'arn
JP6060417B2 (ja) * 2014-07-01 2017-01-18 国立研究開発法人情報通信研究機構 立体ディスプレイ
US20190385697A1 (en) * 2017-02-14 2019-12-19 The Regents Of The University Of Colorado, A Body Corporate Methods for predicting transcription factor activity
GB201912103D0 (en) * 2019-08-22 2019-10-09 Univ Oxford Innovation Ltd Method of haplotyping
EP4176078A4 (fr) * 2020-07-06 2024-07-24 Arpeggio Biosciences, Inc. Systèmes, méthodes et applications pour une liaison fonctionnelle de positions chromosomiques
CN117012278B (zh) * 2023-08-08 2025-05-16 中国医学科学院肿瘤医院 一种获得组织空间转录本信息的方法、装置及电子设备

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WO2012074855A2 (fr) 2012-06-07
WO2012074855A3 (fr) 2013-04-04
US9284604B2 (en) 2016-03-15
EP2643484A4 (fr) 2014-04-16

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